Myelin phagocytosis and remyelination of macrophage-enriched central nervous system aggregate cultures.

An increased level of myelin basic protein (MBP) degradation peptide 80-89, representative of myelin breakdown, is detected in myelinating foetal rat brain aggregate cultures supplemented with peritoneal macrophages at a time coinciding with the onset of myelination. During the period of myelination, the proportion of activated macrophages/microglia in the aggregates decreases, accompanied by a reduction in the content of MBP degradation products. During the recovery period following a demyelinating episode, the rate of MBP synthesis in antibody-treated standard aggregates was greater than in their medium controls. However, the rate of MBP accumulation was not as efficient in macrophage-enriched aggregates and was associated with persistently raised MBP peptide levels. Thus, as occurs in multiple sclerosis lesions, attempts at remyelination appear to be counterbalanced by macrophage-mediated demyelination, with the continued presence of degraded myelin rendering a local environment that is not fully conducive to remyelination.

Temporal analysis of growth factor mRNA expression in myelinating rat brain aggregate cultures: increments in CNTF, FGF-2, IGF-I, and PDGF-AA mRNA are induced by antibody-mediated demyelination.

Myelinogenesis in rat brain aggregate cultures is associated with a pattern of growth factor mRNA expression comparable to that of the developing brain. The rate of increase in platelet-derived growth factor-AA (PDGF-AA) expression was greatest just before the detection of myelin basic protein (MBP) mRNA in the cultures and remained high thereafter, consistent with in vivo observations. Levels of fibroblast growth factor-2 (FGF-2) and of ciliary neurotrophic factor (CNTF) mRNA increased continuously over the period of MBP accumulation. High rates of transforming growth factor beta1 (TGF-beta1), insulin-like growth factor-I (IGF-I), and neurotrophin-3 (NT-3) expression at early time points during the culture gradually decreased over time, indicative of a key regulatory role during oligodendrocyte development. The addition of demyelinative anti-myelin oligodendrocyte glycoprotein (anti-MOG) antibody resulted in a significant increase in MBP peptide fragments with a C-terminus at phenylalanine 89 indicating proteolytic breakdown of MBP after myelin phagocytosis. Immediately after antibody treatment the expression of CNTF mRNA was significantly increased, compared with controls, while that of FGF-2 and IGF-I, and of PDGF-AA peaked during the early and later stages of recovery respectively. Thus, specific growth factors combine to regulate myelination and remyelination in the aggregates; these data have implications for demyelinating disease in which protective growth factor secretion may be central to regeneration.

Myelin basic protein isoforms in myelinating and remyelinating rat brain aggregate cultures.

Recent evidence suggests that myelin basic protein (MBP) exon-2-containing isoforms play a significant role in the onset of myelination because they are more abundant during early development. The pattern of expression of MBP exon-2-containing isoforms was studied in rat brain aggregate cultures during myelination to draw comparisons with the developing brain and at remyelination after demyelinative treatment. The pattern of MBP isoform expression in the aggregate cultures was found to be similar to that of the brain and was recapitulated after demyelination with antimyelin antibodies. Macrophage enrichment, resulting in increased accumulation of total MBP in the cultures, did not alter the isoform distribution. Both control and enriched cultures expressed a 16-kDa protein (26+/-9.8% of total MBP for control samples) that reacted with MBP antisera at the onset of myelination (day in vitro 14) but was barely detectable by day in vitro 21. The expression of this protein, also present in postnatal day 6 rat brain but no longer by day 11, has been predicted by reverse transcription polymerase chain reaction in embryonic mouse brain. The results of the present study reinforce the value of the aggregate culture system as a versatile yet accurate model of myelination and remyelination.

Macrophages in CNS remyelination: friend or foe?

Hematogenous macrophages and resident brain microglia are agents of demyelination in multiple sclerosis (MS) and paradoxically may also participate in remyelination. In vitro studies have shown that macrophage enrichment of aggregate brain cultures promotes myelination per se and enhances the capacity to remyelinate following a demyelinating episode. It has been hypothesized that remyelination in MS is implemented by surviving dedifferentiated oligodendrocytes or by newly recruited progenitors that migrate, proliferate and synthesize myelin in response to signalling molecules in the local environment. We postulate that macrophage-derived cytokines or growth factors may directly or indirectly promote oligodendroglial proliferation and differentiation, contributing to myelin repair in inflammatory demyelinating disease.

Myelination and remyelination of aggregate rat brain cell cultures enriched with macrophages.

We reported previously that accumulation of myelin basic protein (MBP) in foetal brain aggregate cultures is enhanced by supplementation with peritoneal macrophages. The present study demonstrates that the rate of MBP accumulation in macrophage-enriched cultures continues to increase over time unaccompanied by a matching increase in the oligodendrocyte marker cyclic nucleotide phosphodiesterase, while that of control cultures reaches a plateau. These observations are supported by electron microscopic evidence of cumulative numbers of myelinated axons in the aggregates over time and by enhanced expression of myelin protein genes in macrophage-enriched relative to control cultures. Aggregates demyelinate following short-term exposure to cytokines and antimyelin oligodendrocyte glycoprotein antibody, and MBP synthesis resumes following removal of demyelinating agents. Supplementation of cultures with macrophages influences the degree of myelin breakdown and remyelination, drawing attention to the role that macrophage-derived growth factors may play in myelinogenesis and myelin repair in inflammatory demyelinating disease.